CZ295933B6 - Lawsonia intracellularis cultivation process and anti-lawsonia intracellularis vaccine - Google Patents

Abstract

In the present invention, there is disclosed a method for large scale cultivation and attenuation of L. Intracellularis bacteria by inoculating cells with L. Intracellularis bacteria to infect the cells, incubating the infected cells at a temperature ranging from 36 to 38 degC in a reduced oxygen concentration and maintaining the infected cells in suspension. Anti-L. Intracellularis vaccines are prepared from cultures grown in suspension. Disclosed are also a method for determining the presence of antibodies, intracellularis bacteria and attenuated variant thereof.

Description

Lawsonia intracellularis culture method and vaccine

Technical field

The present invention relates to vaccines against Lawsonia intracellularis and a method of protecting against this bacterium and diagnosing an infection caused by the bacterium. The results and methods of the invention are partially achievable due to the improved method for large-scale cultivation of L. intracellularis that has been discovered.

BACKGROUND OF THE INVENTION

In fact, L. intracellularis, the cause of porcine proliferative enteropathy ("PPE"), affects all animals, including humans, rabbits, ferrets, hamsters, foxes, horses, and other animals as diverse as ostrich and emu.

L. intracellularis is a significant cause of losses in pig breeding. Estimates of the incidence and emergence of PPE in the US are around 20% of pig farms with annual estimated losses of $ 20 million.

A common feature of PPE is the occurrence of intracytoplasmic curved rods not bound to the membrane within enterocytes in the affected parts of the intestine. Bacteria associated with PPE were referred to as "Campylobacter-like organisms". S. McOrist et al., Vet. Pathol, Vol. 26, 26064 (1989). Thereafter, the bacteria causing the disease were identified as a new taxonomic genus and species, in the scientific language referred to as the Ileal symbiont (IS) intracellularis. C. Gebhart et al., Int. J. of Systemic Bacteriology, Vol. 43, No. 3, 535-38 (1993). More recently, this new bacterium has been given the taxonomic name Lawsonia (L.) intracellularis. S. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4, 820-25 (1995). These three names have been used interchangeably to refer to the same organism, which is further identified and described herein. The present inventors have sought to use the taxonomic name, L. intracellularis, to discuss the present invention.

L. intracellularis is an obligate intracellular bacterium that cannot be cultured by normal bacteriological methods on a conventional cell-free medium and has been thought to require attached epithelial cells to grow. S. McOrist et al., Infection and Immunity, Vol. 61, No. 10, 4286-92 (1993) and G. Lawson et al., J. of Clinical Microbiology, Vol. 31, No. 5, 1136-42 (1993) discuss culturing L. intracellularis using rat intestinal epithelial cell monolayers IEC-18 in conventional tissue culture flasks. Furthermore, H. Stills, Infection and Immunity, Vol. 59, No. 9, 3227-36 (1991) discusses the use of Intestine 407 human embryonic intestinal cell monolayers and guinea pig gut adenocarcinoma monolayers GPC-16 in conventional tissue culture flasks. These prior art cultivation methods are labor intensive and not suitable for scale-up.

Obtaining new information on L. intracellularis infection and treatment and effective disease control was severely hampered by the demanding growth demands of Z. intracellularis in in vitro cultures. Therefore, there is a need for an improved method of cultivating Z. intracellularis. There is also a need for vaccines against Z. intracellularis and effective tools for diagnosing Z. intracellularis infection.

SUMMARY OF THE INVENTION

It is an object of the invention to provide anti-Z vaccines. intracellularis.

Another object of the invention is to provide methods for detecting the presence of antibodies against Z. intracellularis in biological samples.

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Another object is to provide an improved culture method that allows large scale cultivation of L. intracellularis for the production of vaccines and diagnostic devices.

To achieve this and other objects, and in accordance with the purpose of the invention as described herein, the present invention provides a method of culturing Z. intracellularis and obtaining a large number of bacteria in this manner.

According to this method, L. intracellularis bacteria are incubated at an oxygen concentration of from about 0% to about 18% while mixing the bacteria, wherein the L. intracellularis bacteria are maintained by stirring in suspension.

According to another embodiment, there is provided a method of culturing L. intracellularis by inoculating a monolayer of HEp-2, McCoys, or IEC-18 cells that is about 30% confluent with an inoculum containing L. intracellularis to infect bacterial cells. Infected cells are then incubated at about 36 to about 38 ° C at an oxygen concentration of about 0% to about 8.0% until confluent cell growth is achieved. The infected cells and growth medium are then placed in a fermenter, bioreactor, spinner flask or other container suitable for keeping the cells in suspension. Infected cells are incubated to cultivate L. intracellularis cells while mixing the cells while keeping the infected cells in suspension. A portion of the cultured L. intracellularis is then passaged into fresh culture to increase the production of L. intracellularis.

The invention provides L. intracellularis vaccines and a method of making L. intracellularis vaccines. Avirulent L. intracellularis bacteria are obtained by passage of cultured L. intracellularis bacteria with a sufficient number of passages and selection for an attenuated strain or exposure of the cultured bacterium to attenuating chemicals. L. intracellularis killed vaccines are also prepared using the culture of the invention. According to a particularly preferred embodiment, the bacteria are continuously cultured for at least 6 to 8 months at a passage of at least 7 to 12 times to obtain an attenuated strain suitable for use as a vaccine. The attenuated bacterium is then mixed with a pharmaceutically acceptable carrier and administered in an effective amount to a human or animal to elicit an immunological response. The currently preferred attenuated strain (N343NP40wk) was deposited in the American Type Culture Collection.

The invention also provides the use of L. intracellularis bacteria for determining the presence of antibodies that specifically react with L. intracellularis in a biological sample by isolating at least a portion of cultured L. intracellularis bacteria by contacting the biological sample from an animal with isolated L. intracellularis bacteria or components thereof. conditions under which the antibody present in the biological sample reacts with L. intracellularis or a component thereof, and determining whether or not an antibody-antigen reaction has occurred.

Other features and advantages of the invention will be set forth in the following description and will be apparent from the description or the appended examples.

As used in the present application, the term "L. intracellularis' means an intracellular curved gram-negative bacterium described in detail in C. Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-38 (1993) and S. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4, 820-25 (1995) and include, but are not limited to, bacteria deposited as ATCC 55672 in the American Type Culture Collection, Rockwille, MD; bacteria deposited as NCTC 12656 and 12657 at the National Collection of Type Cultures, Colindale, London; disease causing bacteria that can be obtained from pigs infected with PPE or other animals worldwide, using the knowledge and experience of the art and the present invention; and variants or mutations of any of the above bacteria, obtained artificially or spontaneously.

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As used herein, the term "attenuated strain" means any L. intracellularis strain prepared by culturing the invention and passaging the invention to achieve avirulence while maintaining immunogenic properties when administered to a host. As shown below, the methods of the invention were cultured and attenuated by different strains of L. intracellularis to obtain attenuated immunogenic strains effective as vaccines in pigs and other animals susceptible to L. intracellularis infection.

The attenuated strains of the invention are expected to be used as immunogens in antimicrobial vaccines for animals, including birds, fish, cattle, pigs, horses, generally mammals and primates, and humans. These vaccines can be prepared by methods known to those skilled in the art according to the methods used in the present invention. Such a vaccine would comprise an immunologically effective amount of the attenuated strain in a pharmaceutically acceptable carrier. The vaccine would be administered in one or more doses. The immunologically effective amount can be determined without further experimentation by means known in the art and by the compositions of the present invention. The amount of avirulent bacteria should be sufficient to stimulate an immunological response in a disease-susceptible organism while still remaining avirulent. This will depend on the particular animal, bacterium and disease being treated. The recommended dose for administration to the susceptible organism is preferably about 10 ³ to 10 ⁹ bacteria / kg body weight, and preferably about 10 ⁵ to 10 ⁷ bacteria / kg body weight. Those skilled in the art are familiar with useful carriers, including stabilizers and diluents. The vaccine may also contain a suitable adjuvant. The vaccines of the invention may be used in combination with other vaccines, for example, as a diluent of another lyophilized vaccine, or may be combined with another vaccine prior to lyophilization. The vaccine formulations may also be in the dried state, for example by lyophilization, especially for storage reasons or for further addition to liquid vaccines.

The invention is useful for inducing an immunological response to a virulent wild-type L. intracellularis bacterium in a human or animal in order to achieve protection of the host against the bacterium. The induction comprises administering to the host an immunologically effective amount of an attenuated bacterium or killed bacterium of the invention and preferably administering to the host a vaccine of the invention.

As used herein, the term "large-scale cultivation" means a range of cultivation of L. intracellularis greater than about 2.0 to 3.0 L and includes production on a volume scale of 100 L or more. "Cultivation" as used herein means a method of inducing L. intracellularis growth, reproduction and / or proliferation.

In carrying out the culture method of the invention, the cell culture may first be inoculated with an inoculum containing L. intracellularis so as to infect the cells with bacteria. A variety of cell lines can be used in the practice of the invention including, but not limited to, IEC-8 (ATCC 1589) - rat intestinal epithelial cells, HEp-2 (ATCC 23) - human epidermis carcinoma cells, McCoys (ATCC 1696) - mouse (unspecified) cells. MDCK (ATTC 34) - canine kidney cells Madin-Darby, BGMK (Biowhittaker # 71-176) - buffalo green monkey kidney cells and porcine intestinal epithelial cells. Preferred tissue culture cells are HEp-2, McCoys or IEC-18. Alternatively, the bacteria may be cultured in a cell-free system as long as the bacteria are maintained at a suitable dissolved oxygen concentration of the invention.

If a cell culture is used, the culture cells are preferably in the form of a monolayer prior to inoculation, but need not be so. To form a monolayer, cells can be seeded into conventional bottles. Each flask is typically seeded onto a 25 cm ² flask with a number of cells ranging between about 1 x 10 ⁵ to about 10 x 10 ⁵ mixed with the growth medium. The growth medium may be any cell culture medium that contains a nitrogen source, necessary growth factors for the selected cultured cells, and a carbon source such as glucose or lactose. The preferred medium is DMEM with 2-5% fetal bovine serum, although other commercially available media may be used with good results.

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It has been found by the inventors that the success rate of L. intracellularis cultivation is increased by keeping tissue culture cells at a constant growth stage. Therefore, the tissue culture monolayer should be in a state of about 20% to about 50% confluency at the time of inoculation. Preferably, at the time of inoculation, the cell culture should be in a state of about 30% to about 40% confluency, most preferably about 30% confluency.

The inoculum may be a pure culture of L. intracellularis, obtained, for example, from a strain having deposit number ATCC 44672, NCTC 12656 or 12657, or from infected pigs or other animals using said isolation and purification procedures.

According to one embodiment of the invention, the inoculum for practicing the invention is an intestinal homogenate prepared by scraping the mucosa of a pig or other PPE infected animal. When preparing the intestinal homogenate, the intestinal sections selected for culture should show significant changes with macroscopic thickening of the intestinal wall. For bacterial fragility, samples should be stored at -70 ° C as soon as possible after necropsy. To suppress contaminating bacteria while maintaining the growth of L. intracellularis, antibiotics to which L. intracellularis is resistant, such as vancomycin, amphotericin B or members of the aminoglycoside family of antibiotics including gentamycin and neomycin, are preferably added to the inoculum. Whether the inoculum is a pure culture or an intestinal homogenate, the inoculation of the cell culture can be carried out by various methods and procedures known in the art according to the invention.

The bacteria and / or inoculated cell cultures are then incubated at a reduced concentration of dissolved oxygen. At a dissolved oxygen concentration of greater than 18%, L. intracellularis growth is no longer optimal, and growth concentrations may occur at oxygen concentrations outside this range. Preferably, seeded cell cultures are incubated at a dissolved oxygen concentration in the range of about 0% to about 10%. Most preferably, the cells are incubated at an oxygen concentration in the range of about 0% to about 8% with the most preferred oxygen concentration in the range of about 0% to about 3.0%.

The correct concentration of carbon dioxide is also important for the correct growth of L. intracellularis. Growth deterioration or even growth arrest occurs outside the carbon dioxide concentration in the range of greater than 10% and less than 4%. Preferably, the concentration of carbon dioxide is in the range of about 6% to about 9%, and most preferably about 8.8%.

In addition, the cells are preferably incubated at a hydrogen concentration of about 73% to about 94%. Nitrogen may be used instead of some or all of the hydrogen present. According to a particularly preferred embodiment, the cells are incubated at a gas composition of about 0 to 8.0% O ₂ , about 8.8% CO _2, and about 83.2% H ₂ .

The inoculated cells may be incubated in an incubator allowing the introduction of two gases, or another space allowing cultivation in an atmosphere of gases containing appropriate concentrations of oxygen and carbon dioxide and allowing the cells to be suspended during incubation. The culture chamber should include a means for keeping the inoculated cells in suspension, a gas monitor, and a gas source to maintain the appropriate gas concentration. The incubation temperature should be in the range of 30 ° C to 45 ° C, and more preferably in the range of about 36 ° C to about 38 ° C. Most preferably, the temperature will be about 37 ° C. The necessary equipment for culturing and attenuating the cells of the invention will be available to those skilled in the art using the methods of the invention. An example of equipment suitable for practicing the present invention is a dual-gas incubator, for example, Model 480 of Lab-Line, Melrose Park, Illinois, in conjunction with rotary flasks to keep cells in suspension. Preferred equipment includes a fermenter, bioreactor or rotary shaker containing at least about 2 L of medium and capable of keeping the cell culture in suspension by bubbling gases at a suitable concentration or by mechanical stirring and continuously measuring dissolved oxygen concentrations in the medium. Suitable fermenters and bioreactors for this purpose are manufactured by New Brunswick, Braun et al.

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The maintenance of the inoculated cells during incubation in suspension is achieved by increasing the exposure of each cell to the growth medium and the correct mixture of oxygen and carbon dioxide to maximize cell growth and thereby L. intracellularis. The cell culture may be mixed or suspended in a variety of methods known in the art, including, for example, culture flasks, spinner flasks, membrane cultures, and rolling flasks. The cells may be kept in suspension during incubation by incubating the cells in a rotating flask inside a bi-gas incubator or similar device. The term "spinner flask" as used herein refers to a bottle or other container containing blades, a stirrer or other means for mixing the culture and keeping the cells present in suspension.

In a particularly preferred embodiment of the invention, the inoculated cells are incubated until confluence is reached and then placed in a stirred bottle containing the growth medium and incubated in an incubator while the bottle is rotated. Preferably, the inoculated cells are scraped into a rotary bottle. This can be achieved by a variety of methods known in the art, for example using a cell scraper to release the cells. Once the cells are introduced into the stirred bottle, its paddle typically rotates at a rate of about 30 to about 60 rpm to keep the infected cells in suspension.

A portion of the cultured L. intracellularis is then passaged into fresh cell culture to increase L. intracellularis production. The term "passage" or variation thereof means a method of transferring a portion of cultured L. intracellularis cells to a fresh cell culture in order to infect fresh bacteria cells. The term "fresh" as used herein means cells that have not yet been infected with L. intracellularis cells. Preferably, such cells are on average no more than about one day old.

Passage of L. intracellularis in suspension cultures can be accomplished by removing part of the original culture and adding this part to a new flask containing fresh cell culture cells. If the original culture has a high number of bacteria per ml, for example greater than about 10 ⁴ bacteria / ml, it is preferred to add between about 1-10% (v / v) of the culture from the infected flask to a new flask containing fresh cells. This is preferably done when 50-100% of the cells are infected. If less than 50% of the cells are infected, passage is preferably performed by dividing the culture in a ratio of 1: 2 into a new flask and increasing the volume by adding fresh medium. In any case, unlike the passage of cultures in prior art monolayers, cell lysis and other steps are not required.

After sufficient growth of the cell culture and subsequent infection by L. intracellularis with an infection rate greater than about 70% as determined by IFA, TCID ₅₀ or other comparable method, at least a portion of the cultured L. intracellularis bacteria is isolated. Isolation can be carried out by separating the bacteria from the suspension in various known ways. Preferably, L. intracellularis bacteria are isolated by centrifuging part or all of the suspension volume to obtain biomass of cultured cells, resuspending the recovered cell biomass and lysing the infected cells. Typically, at least a portion of the medium is centrifuged for about 20 minutes at about 3000 xg to recover cell and bacterial biomass. The biomass can then be resuspended in sucrose-phosphate-glutamate (SPG) solution and cell lysis can be accomplished by approximately four times passing through a 25 gauge needle. If further purification is required, samples can be centrifuged to remove cell nuclei and cell portions for approximately 5 min at 145 The supernatant can then be centrifuged for approximately 20 minutes at approximately 3000 xg and the resulting biomass resuspended in a suitable diluent such as SPG with fetal bovine serum (to prepare isolated bacteria suitable for freezing as an inoculum) or growth medium (to prepare isolated bacteria more suitable). for passage into fresh cells.

As mentioned previously, efficient growth of L. intracellularis for large-scale production can be enhanced by maintaining the active growth of tissue culture cells. In the case of monolayers, the cell division rate is significantly reduced when confluence is reached. Attempts to grow L. intracellularis on monolayer tissue cultures have had limited success and could not be scaled up. However, the use of the cultures in suspension will allow the cells to be maintained in active growth and allow for the passage of cells

-5E 295933 B6 normal culture expansion and scaling. Using a fermenter and at a dissolved oxygen concentration of between about 0 to 3% as explained above, the inventors achieved growth of up to 10 ⁸ bacteria per ml. It has also been possible to maintain the cultured bacteria in active growth for many months and it is expected that the bacteria could grow permanently in this way.

In the period prior to the present invention, it was generally believed that cells must be attached to the surface in order to be infected with L. intracellularis. The cell suspensions of the present invention are therefore unique and contradictory to this theory. When McCoys or IEC-18 cells are used, it is preferred to add gelatin, agarose, collagen, acrylamide or silica beads such as Cultisphere-G porous microcarriers manufactured by HyClone Laboratories, Logan, UT along with the growth medium. However, HEp-2 cells do not require microcarriers for cultivation according to the invention. This is a particularly advantageous and economical method of large-scale cultivation.

For culture purposes, in the case of cultures with HEp-2 cells, preferably 25 to 50% of the culture is removed at weekly intervals and replaced with fresh medium. In the case of cell cultures with microcarriers or beads, preferably 25 to 50% of the culture is removed once or twice a week and replaced with fresh microcarriers and beads and fresh medium. For scale-up purposes, an additional 25-50% medium or microcarrier medium may be added to the culture.

Depending on the rate at which the cells of the culture are infected, the passage of fresh cells usually takes place approximately every two to every five weeks. Assuming that the cell culture cells are at least 70% infected within two to three weeks, a preferred passage term occurs approximately every three to four weeks.

The present invention also provides vaccines and methods for producing L. intracellularis vaccines. According to a particularly preferred embodiment, after keeping the infected cells in suspension for a longer period (e.g. 6-8 months), at least a portion of the cultured L. intracellularis bacteria is isolated and monitored for potential attenuation. Such monitoring is preferably performed by a culture administration test to a host animal or animal model to select an attenuated strain. Such attenuated strains are used as vaccines according to the methods of the present invention. The attenuated L. intracellularis vaccines of the present invention have been shown to be effective against L. intracellularis infection in a number of animals and are expected to be successful in humans as well.

The present invention allows a rapid increase in culture, i.e. an increase in yield of 100 to 1000 times and a reduction in costs. As a result, the excess amount of L. intracellularis produced by the cultivation method of the invention is readily attenuated for vaccine production purposes. Weakening is difficult for monolayer cultures because of the low yield of bacteria produced by conventional monolayer culture. In contrast, the method of growing L. intracellularis of the present invention greatly increases the ease, speed and number of bacteria available for this purpose. The larger the number of cells and the cell divisions involved, the greater the occurrence of mutations that are beneficial for vaccine development. Growth in the suspensions according to the invention also increases the expression of important iminogens, driven by environmental-influenced genes and their expression products.

The resulting attenuated strains can be cultured in a tissue culture monolayer as described in Example 1 below, but preferably the culture is performed in the suspension cultures of the invention. Other means of attenuation may include chemical attenuation using, for example, N-methylnitrosoguanidine and other means known in the art. An attenuated L. intracellularis bacterium, obtained either by multiple passages or by chemical means, is produced and selected for vaccine preparation.

According to one embodiment of the vaccine of the invention, the antigen is isolated by centrifugation or microfiltration as described above. The antigen is then standardized to a defined concentration based on the optimal immunological response of the host animal as determined by dose titration in the species of animal host. The bacterium can be inactivated by prolonged exposure, for example one week,

The concentration of ambient oxygen or using 0.3% formaldehyde or other inactivation agent suitable for the preparation of the killed vaccine. The antigen is then introduced into a suitable adjuvant, such as aluminum hydroxide or mineral oil, to achieve an enhanced immunological response. The antigen is then used to vaccinate the host by intramuscular or subcutaneous injection, in the case of pigs of approximately three to four weeks of age and, if necessary, with a booster dose.

According to a particularly preferred method of producing the vaccine of the invention using the culture methods described above, it is also possible to perform serial passage of the bacteria to induce and select an attenuated avirulent live culture. The culture is tested in an animal host (after approximately at least 6-8 months of growth in suspension culture) for signs of attenuation. The culture is isolated as described above and diluted. For example, pigs are vaccinated by mouth of 1 x 10 ⁵ to 1 x 10 ⁶ bacteria. Approximately 28 days after vaccination, pigs are orally inoculated with approximately 1 x 10 ⁷ microorganisms after a smaller number of passages (approximately 30 to 45 days old) of virulent L. intracellularis culture. Infected animals are necropsied 21 days after administration and the small intestine is examined for large-scale and microscopic lesions. It is also possible to perform PCR and assay with fluorescent antibodies. Approximately 80% of the control animals will have major or microscopic lesions and a positive test for L. intracellularis in intestinal mucosal cells using either PCR or FA. Vaccinated animals will have mucosal surfaces normal in histological observation and will be negative when tested by PCR.

Typically, an attenuated immunogenic L. intracellularis strain is produced after continuous culture for at least 150 to 250 days, at which time the culture is passaged at least about seven to about twelve times. When the monitoring and selection method of the present invention is used, further attenuated cultures can be produced, thereby altering these numbers.

A vaccine is then prepared which contains an immunologically effective amount of attenuated L. intracellularis in a pharmaceutically acceptable carrier. The combined immunogen and carrier can be an aqueous solution, emulsion or suspension. The immunologically effective amount can be determined by methods known in the art without further experimentation based on the data presented herein. The amount of immunogen will generally be between 50 and 500 µg and preferably between 10 ⁷ and 10 ⁹ TCfD ₅₀ using purified bacteria.

The vaccines of the invention are usually administered to susceptible animals, particularly pigs, in one or more doses. The live or killed vaccine may be administered once or twice at two-week intervals. For attenuated live vaccines, a single dose is preferred. Preferred routes of administration in live attenuated strains are oral or intranasal, but intramuscular or subcutaneous injection may also be used. Intramuscular and subcutaneous injections are the most preferred methods for a killed vaccine.

Effective diagnosis of PPE has also been hampered by the time required to cultivate the disease-causing bacteria. As a result of the present invention, it is now possible to develop diagnostic means for rapidly and accurately testing the presence of L. intracellularis in biological samples taken from pigs and other animals susceptible to PPE.

L. intracellularis bacteria grown by the method of the present invention or components derived from such bacteria may be used as an antigen in an ELISA or other immunoassay, such as an immunofluorescence antibody test ("IFA") to detect antibodies against L. intracellularis in serum and other body fluids animals suspected or infected with bacteria. A preferred immunoassay is currently the IFA described in the example below. Alternatively, bacteria grown by the method of the invention in a western blot test may also be used.

A preferred ELISA protocol according to this embodiment of the invention is as follows:

1. Add 0.1 ml / well of antigen diluted in coating solution. Incubate for 18 h at 4 ° C.

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2. Wash three times with PBS.

3. Add 0.25 ml blocking buffer to each well of the plate. Incubate for 1 to 2 hours at 37 ° C.

4. Wash three times with wash buffer.

5. Dilute serum in blocking buffer and add 0.1 ml to the first wells of the plate. Serial dilutions of 1: 2 are made across the plate. Incubation is performed at 37 ° C for 1 h.

6. Wash three to five times with wash buffer.

7. Dilute conjugate in blocking buffer, add 0.1 ml to wells of plate and incubate for 1 hour at 37 ° C.

8. Wash three to five times with wash buffer.

9. Add substrate.

12. Measure the absorbance using a spectrophotometer.

13. Wells without antigen addition are used as blank.

14. Swine serum can also be used as positive and negative controls in each test.

A preferred Western blot protocol is as follows:

1. Electrophoresis of 12% SDS-PAGE antigen and transfer to a nitrocellulose membrane.

2. Place the membrane in blocking buffer for 2 hours.

3. Remove blocking buffer and rinse with PBS for 1 min.

4. The serum is diluted with blocking buffer and added to the membrane. Incubation is carried out for 2 hours at room temperature.

5. Wash three times with wash buffer (5 min for each wash).

6. Dilute conjugate with blocking buffer and add to membrane. Incubate for 1 hour at room temperature.

7. Wash three times with wash buffer.

8. Substrate is added for 10 min or until strong bands are formed.

9. Rinse with PBS.

10. The membrane is air-dried and stored in the dark.

The present invention is further described by the following examples, which are intended to be illustrative only and are not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Isolation of L. intracellularis from intestines of American swine with proliferative enteropathy of swine

Materials and methods:

Selection of samples for inoculation:

Sample N24912 was obtained from a lowland farm, where 15 out of 300 five-month-old pigs were observed to have persistent bloody stools regardless of penicillin treatment. After porcine necropsy

Thickened mucosa was observed in the intestine (ileum). Histopathological examination with silver staining showed the presence of curved intracellular bacteria and enterocytic hyperplasia in crypts, confirming the diagnosis of PPE. Sample N72994 was obtained from 1.5-year-old sows after the second litter on a farm in Minnesota. Breeding sizes ranged from 70 to 80 sows and no antibiotic administration was observed. Thickening of the ileum with bleeding was found after necropsy. Staining of the mucosa according to Giminez showed a large number of curved bacteria. Sample NI01494 was obtained from 12 week old pigs from an Indian farm with 600 breeding sows. The pigs were treated with Tylan injections after the onset of bloody diarrhea, but died soon after treatment.

Preparation of inoculum of porcine-derived bacteria:

Intestinal samples were stored at -70 ° C. The intestines were opened and washed with phosphate buffered saline (PBS). One-gram mucosal samples were scraped into sodium potassium-glutamate buffer (SPG) and homogenized for 30 sec with 4.0 ml of 1% trypsin (JRH Biosciences, Lenexa, KS) in SPG. The suspensions were incubated for 35 min at 37 ° C. 10 ml of SPG / 10% fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS) was added and the samples were milled in a tissue homogenizer for 1 minute. 10 mL of SPG / 10% (FCS) was added and the suspension was once filtered with filter paper (Whatman 113V; Whatman Labsales, Hillsboro, OR) and then sequentially through 5.0, 1.0 and 0.65 µm membrane filters. The filtrates were aliquoted into 1.0 ml and stored frozen at -70 ° C. The mucosa was spread on glass for staining according to Giminez. Separate smears of the filtrates were stained with IFA using a specific monoclonal antibody against L. intracellularis (S. McOrist et al., Vet. Rec. 121: 421-422 (1987)).

Cell culture:

IEC-18 cells (rat intestinal epithelial cells, ATCC CRL 1589) were grown in DMEM (JRH Biosciences, Lenexa, KS) with glutamine and 10% FCS with weekly trypsin passage. Mono cell layers were grown at 37 ° C in air with 5% CO ₂ .

Cell culture infections:

IEC-18 cells were seeded at a concentration of 1.25 x 10 ⁵ cells in 25 cm ² flasks and in comparable quantities in chamberslides (Nunc, Inc., Naperville, IL). The cells were incubated for 24 hours and then the medium was removed. Frozen porcine bacterial isolates were rapidly thawed and diluted in DMEM / 7% FCS with vancomycin (100 µg / ml) and amphotericin B (2.0 µg / ml) at 1.0 ml homogenate per 15 ml medium and added to the monolayers. Monolayers and bacterial suspensions were centrifuged for 30 min at 2000 g and transferred to anaerobic incubators. The incubators were evacuated and the gas was replaced with hydrogen and carbon dioxide to form a mixture of 8.0% O ₂ , 10% CO ₂ and 82% H ₂ . The cultures were incubated for 3 hours at 37 ° C, then DMEM / 7% FCS with L-glutamine, vancomycin (100 µg / ml), neomycin (50 µg / l) and amphotericin B (2.0 µg / ml) were added. Cultures were changed in anaerobic incubation flasks and incubated for 6 days with medium change every two days.

Passage of L. intracellularis.

L. intracellularis bacteria were cell lysed using potassium chloride as previously described in: G. Lawson et al., J. Clin. Microbiol., 31: 1136-1142 (1993) and then added to fresh IEC-18 cell monolayers. Media was cast from monolayers, 0.1% KCl was added and cells were incubated for 10 min at 37 ° C. KCl was removed and SPG / 10% FCS was added and the monolayers were mechanically released with a scraper. Cells were lysed by passing three times through a 21 gauge syringe. The cell nuclei were removed by centrifugation at 100 xg for 5 min and the bacterial suspension in the supernatant added to fresh 1d IEC-18 cell monolayers.

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Monitoring of cell culture infection:

The infection was monitored by fixing the cells on the slides with cold acetone / methanol for 5 minutes. Staining was performed by immunofluorescence and immunoperoxidase methods. Both methods used murine monoclonal antibody (as described in: S. McOrist et al., Vet. Rec. 121: 421-422 (1987)) as the primary antibody and either the anti-mouse immunoglobulin G-fluorochrome conjugate (fluoresein isothiocyanate; Organon Teknika Corporation, Durham , NC), or peroxidase conjugate (goat anti-mouse immunoglobulin G; Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) Quantification of bacteria was performed by counting specifically stained bacteria inside cells on each slide.

Polymerase chain reaction:

A sample of inoculum and passaged bacteria was used as a template DNA in PCR using a method for preparing samples, primers and cycling parameters as described by Jones et al., J. Clin. Microbiol., 31: 2611-2615 (1993) and McOrist et al., Vet. Microbiol. 1-8 (1994). The cycling parameters were for the first cycle: 93 ° C 5 minutes, 55 ° C 45 s and 72 ° C 45 s. 33 cycles were performed at the above temperatures for 45 s per temperature and one cycle 93 ° C 45 s, 55 ° C 45 to 72 ° C 2 min. Only positive inoculum was used to inoculate IEC-18 cells. PCR was also performed to monitor passaged material to confirm infections. DNA generated by PCR was sent to Iowa State University's Nucleic Acid Facility laboratory for sequencing. The sequencing results were compared to sequences produced by: Gary F. Jones, Ph.D., University of Minnesota, Minneapolis, MN (June 1993).

Results:

Selection of inoculum samples:

Pig numbers N24912 and N72994 had severe PPE with bloody intestine and mucosal thickening. Pig number NI01494 had severe PPE and severe bleeding which resulted in a large blood clot in the intestine. Staining of mucosal smears according to Giminez showed a large number of curved S-shaped bacteria. IFA staining showed a large number of brightly fluorescing bacteria in the case of porcine bacterial inoculation.

Monitoring of cell culture infection:

The inoculated monolayers were monitored during the growth cycle by light microscopy, and little morphological changes of the cells were observed. Uninfected monolayers grew under reduced oxygen pressure (8% (¾) and had similar morphology.

Infected cultures stained with immunofluorescence and immunoperoxidase method showed a large number of curved or S-shaped bacteria that were specifically stained and apparently found inside cells. Monolayers did not have a confluent infection. Infected cells were often in close proximity to infected foci of one to ten cells. Severely infected cells (i.e. cells with 30 or more bacteria) were also seen along with cells with less than thirty bacteria. Bacterial counts peaked after approximately 6 days. The infection was dependent on specific growth conditions. The bacteria were successfully passaged by the cell lysis method described above. Centrifugation of the newly inoculated cells was not necessary but increased the number of infected cells. Centrifugation also reduced contamination by allowing infected cells in the antibiotic-free medium after three hours to exchange the medium with the antibiotic medium. Reducing the FCS content from 10% to 7% in the medium was necessary to slow the growth of IEC-18 cells, allowing the bacteria to expand more before cells in the monolayer reached confluency.

Polymerase chain reaction:

PCR of chromosomal DNA generated a 319 bp fragment (including primers) from all isolates. A suitable size fragment was visually compared to a known positive sample generated using PCR by McOrist et al. (1994). Sequence analysis of PCR products

-10GB 295933 B6

N24912, N72994 and N101494 confirmed tight homology (97-99%) to the p78 sequence determined by Jones (1993).

Example 2

L. intracellularis growth in HEp-2 cell suspension cultures

Preparation of intestinal homogenates for inoculum:

Intestinal homogenate was prepared by scraping the mucosa from 6.0 to 8.0 cm ileum from the intestinal samples of Example 1. Trypsin (1%) was added to the scraped mucosa and the samples were briefly homogenized and then incubated for 35 min at 37 ° C. Then 10 ml of SPG / 10% FBS was added and the samples were ground on a tissue homogenizer. An additional 10 mL of SPG / 10% FBS was then added. The homogenates were filtered through a Whatman VI13 filter and then sequentially through 5.0, 1.0 and 0.65 µm filters. Samples were filled in 1 ml aliquots and frozen at -70 ° C.

Cell culture infections:

Method A

Tissue cells were seeded at a concentration of 1 x 10 ⁷ cells in 50 ml DMEM / 10% FBS into a 100 ml rotary flask. The cultures were incubated for 24 h and then vancomycin and fungizone were added. One ampoule of frozen intestinal homogenates was thawed rapidly and diluted into 3.0 ml DMEM / 5% FBS with vancomycin (100 µg / ml) and amphotericin B (2.0 µg / ml). The sample was filtered through a 0.65 µm filter and added to the bottle. The culture was placed in a gas incubator, evacuated and the gas replaced with a mixture of hydrogen and carbon dioxide to give a mixture of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . The cultures were incubated for 3 h at 37 ° C and then neomycin and gentamycin were added. The medium was changed after 24 hours using DMEM / 5% FBS medium with L-glutamine, vancomycin (100 pg / ml), neomycin (50 pg / l), gentamycin (50 pg / l) and amphotericin B (2.0 pg) / ml).

Method B

Two conventional 25 cm ² flasks were seeded with 1.25 x 10 ⁵ HEp-2 cells in DMEM / 10% FBS and allowed to grow for 18-24 hours. At the time of inoculation, the cells were in 30% confluency. The inoculum was diluted with DMEM / 5% FBS. If the inoculum comes from intestinal homogenates, the media also contain vancomycin (100 pg / ml) and amphotericin B (2.0 pg / ml). The cultures were placed in a gas incubator, evacuated and the gas was replaced with a mixture of hydrogen and carbon dioxide to achieve a mixture of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . In the cultures, the medium was replaced after 24 hours with new DMEM / 5% FBS medium with L-glutamine, vancomycin (100 mg / ml), neomycin (50 pg / l), gentamycin (50 pg / l) and amphotericin B (2, 0 µg / ml). If the inoculum was a pure culture, no antibiotics were necessary. The cultures were incubated for 6 days or until confluence was reached. Cells were scraped from the flasks and added to a 100 ml spinner flask containing 50 ml DMEM / 5% FBS.

The culture was diluted 1: 2 each week either by harvesting half of the culture and adding fresh medium, or by passage to a larger rotary bottle and adding more medium.

Culture Passage:

The culture was transferred to fresh HEp-2 cells by seeding new HEp-2 cells at a concentration of 1 x 10 ⁷ in DMEM / 5% FBS. The new culture was allowed to incubate overnight with a gas concentration of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . The new culture was then inoculated with the infected culture and incubated at reduced O ₂ concentrations as previously reported. The amount of inoculum was dependent on the degree of infection of the original culture.

-11 CZ 295933 B6

Harvest and storage of cultures:

The cultures were harvested by harvesting the required amount of culture by centrifugation for 20 min at 3000 x g. The biomass was resuspended in sucrose-phosphate-glutamate (SPG) solution and passed through a 25 gauge needle four times. The cultures were aliquoted and frozen at -70 ° C. For further purification, samples were centrifuged at 145 x g for 5 minutes to remove cell nuclei and cell debris. The supernatant was then centrifuged for 20 minutes at 3000 x g. The centrifugate was resuspended in the diluent.

Estimated number of viable Z. intracellularis cells in tissue culture:

Quantification of viable L. intracellularis cells was performed by determining the dose for 50% Tissue Culture Infectious Dose 50 percent (TCID50) by removing 2.0 ml of culture for testing and lysing the cells four times with a 25 gauge needle. diluted 1: 10 in DMEM / 5% FBS containing vancomycin (100 µg / ml) and amphotericin B (2.0 µg / ml) Aliquots of the diluted solutions were added to a 96 well microtiter plate at 0.1 ml / well. The microtiter plates were seeded with 1250 cells / well HEp-2 cells, which were allowed to grow for 18 to 24 hours prior to infection, between three and six wells per dilution, and incubated for 6 days at 8.0% O gas concentration. _2, 8.8% CO ₂ and 83.2% H _2. the cells were fixed with cold 50% acetone and 50% methanol for two minutes. to the wells, 0.03 ml / well of anti-IS L. intracellula ris monoclonal antibody (McOrist, 1994) diluted 1: 2000 in PBS. The plate was incubated for 30 min at 30 ° C and then washed three times with PBS. At 0.03 ml / well, anti-mouse FITC diluted 1: 30 was added and the plate was incubated for 30 minutes at 37 ° C. The plate was then washed three times with distilled water and allowed to dry. The samples were observed under a fluorescence microscope and a TCID of ₅ o / ml was determined.

Results:

TCID ₅₀ results show that the cultures contained up to 1 x 10 ⁶ bacteria / ml. This was achieved within 45 days. At the same time, the culture volume was increased to 3.01.

Example 3

L. intracellularis growth in McCoys cell suspension cultures

Preparation of intestinal homogenates for inoculum:

The intestinal homogenate was prepared as described in Example 2. A sample of L. intracellularis cultured according to the following example was deposited in accordance with the Budapest Agreement on May 19, 1995 at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland USA 20852 under deposit number 55672.

Cell culture infections:

1.25 x 10 ⁵ McCoys cells in DMEM / 10% FBS were seeded in two conventional 25 cm ² flasks and allowed to grow for 18-24 hours. At the time of inoculation, the cells are in a state of 30% confluency. The inoculum was diluted with DMEM / 5% FBS. If the inoculum originated from intestinal homogenates, the medium also contained vancomycin (100 pg / ml) and amphotericin B (2.0 pg / ml). The cultures were placed in a gas incubator, evacuated and the gas was replaced with a mixture of hydrogen and carbon dioxide to produce a mixture of 8.0% O ₂ , 10% CO ₂ and 82% H ₂ . The cultures were incubated for 3 hours at 37 ° C and neomycin and gentamycin were added. The culture medium was replaced after 24 hours with DMEM / 5% FBS with L-glutamine, vancomycin (100 pg / ml), neomycin (50 pg / l), gentamycin (50 pg / l) and amphotericin B (2.0 pg) / ml). If the inoculum came from a pure culture, no antibiotics were needed. Cultures were incubated for 6 days until confluence was reached. Cells were then scraped from the flasks and added to a 100 ml spinner bottle containing 50 ml DMEM / 2%

-12EN 295933 B6

FBS and 0.05 g Cultisphere-G Microcarriers. The bottles were mixed at a speed of 40 to 50 rpm.

The culture was diluted 1: 2 every two to three days either by separating half the culture and adding fresh medium and Cultisphere-G beads, or by transferring the culture to a larger rotary flask and adding additional Cultisphere-G beads medium. The final concentration of beads in the culture was approximately 0.001 g beads / ml.

Culture Passage:

The culture was transferred to fresh McCoys cells by seeding 1 x 10 ⁷ new McCoys cells into DMEM / 2% FBS and 0.05 g Cultisphere-G beads. The new culture was allowed to incubate overnight in an atmosphere of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . The new culture was then inoculated with 25 ml of the infected culture and incubated at a reduced Os concentration as above.

Isolation and storage of cultures:

Cultures were isolated by withdrawing the required amount of culture and centrifuging at 3000 x g for 20 min. The biomass was resuspended in SPG and passed through a 22 gauge needle four times. The cultures were aliquoted and frozen at -70 ° C. For further purification, samples were centrifuged at 145 x g for 5 min to remove beads, cell nuclei, and cell portions. The supernatant was then centrifuged at 3000 xg for 20 min. The biomass was then resuspended in the diluent.

Estimation of viable L. intracellularis cells in tissue culture:

The determination of viable L. intracellularis cells was performed as described in Example 2 using a 22 gauge needle for cell lysis and seeding microtiter plates with McCoys cells at 1250 cells / well.

Results:

TCID ₅₀ results showed that the cultures contained up to 1 x 10 ⁶ bacteria / ml. This was achieved in less than 1 month. During the same period, the culture volume was increased to 3.01.

Example 4

Determination of infectious dose of pure L. intracellularis cultures in host animals

Summary:

The 31 pig study was concluded by infection of normal pigs six weeks old with pure L. intracellularis culture from sample N72994. The pigs were randomly divided into four groups, which were placed separately. Group 1 contained seven pigs and was considered to be a negative control group that received uninfected tissue culture or nothing. Group 2 contained eight pigs and received 10 ⁷ bacteria per pig. Group 3 contained eight pigs and received a dose of 10 ⁶ bacteria / pig. Finally, Group 4 consisted of eight pigs that received 10 ⁵ bacteria / pig.

Faecal swabs were collected on days 0, 7, 14 and 21 and on day 24 for PCR testing. On day 24, pigs were sacrificed and their ileum, jejunum and colon were collected for PCR testing, histopathological examination and EA staining as described above.

Intestinal mucosal PCR testing revealed the presence of Z. intracellularis in 100% of the high dose, 75% of the intermediate dose, and 50% of the low dose. Histopathological results showed an increase in the number of mononuclear cells in the lamina propria and the mucosal layer in 88% for the high dose, 75% for the middle dose and 88% for the low dose. Crypt hyperplasia was observed in 50% of the high dose, 63% in the middle dose, and 50% in the low dose. FA staining revealed L. intracellularis in ileum, jejunum and colon tissue sections in 88% of the high dose cases,

B6% for the middle dose and 63% for the low dose. Control animals were negative for the presence of L. intracellularis in PCR, FA and silver staining.

Thus, pure cultures have been used successfully for infection and for inducing PPE damage. Koch's postulates were accomplished by identifying and isolating L. intracellularis from infected animals.

In the test culture animals, in 100% of the high dose cases, bacteria recovery and silver staining, FA and PCR were confirmed.

Materials and methods:

Inoculum growth:

One normal 75 cm ² flask was seeded with 3.75 x 10 ⁵ HEp-2 cells in DMEM / 10% FBS and allowed to grow for 18-24 hours at 37 ° C and 5% CO ₂ (30% at the time of inoculation) confluence). One vial of N72994 was diluted in 15 ml DMEM / 5% FBS. The culture was placed in a gas incubator, evacuated and the gas replaced with hydrogen and carbon dioxide to give a mixture of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . The culture received fresh DMEM / 5% FBS medium at 24 hours.

The cultures were incubated for 6 days, then scraped from the flasks and added to 100 ml spinner flasks containing 50 ml DMEM / 5% FBS. The volume of the bottle was increased by doubling the volume of the medium at weekly intervals. The culture was grown in a rotary bottle for 3 weeks.

Harvesting of cultures:

The culture was harvested by centrifugation for 20 minutes at 3000 x g. The biomass was resuspended in sucrose-phosphate-glutamate (SPG) solution with 10% FBS and passed through a 25 gauge needle 4 times. The inoculum was diluted to a final SPG / 10% FBS volume and prepared. 1:10 dilution.

Control inoculum consisted of uninfected HEp-2 cells diluted to the same viable cell concentration as the infected culture. Cells were harvested in the same way as the infected culture. Control pigs received a similar cell dose to the high dose group.

Quantification of L. intracellularis'.

Quantitation of viable L. intracellularis was performed by determining the tissue culture infective dose 50% (about ₅ TC1D). This was done by collecting 2 ml culture for testing and lysing the cells four times by passing a 22 gauge needle. The sample was serially diluted 1: 10 with DMEM / 5% FBS containing vancomycin (100 pg / ml) and amphotericin B (2.0 pg / ml). Dilutions were added to a 96 well microtiter plate at 0.1 ml / well. Microtiter plates were inoculated with HEp-2 cells at a concentration of 2500 cells / well and allowed to grow for 18-24 hours before infection. 12 wells were used per dilution. The plate was incubated for 6 days in an atmosphere of 8.0% O ₂ , 8.8% CO ₂ and 83.2% N ₂ . Cells were fixed with cold 50% acetone and 50% methanol for 2 minutes. 0.03 ml / well of antiL monoclonal antibody was added to the wells. intracellularis (McOrist, 1987) diluted 1: 2000 with PBS. The plate was incubated at 37 ° C for 30 min and then washed three times with PBS. At a concentration of 0.03 ml / well, anti-mouse FITC diluted 1: 30 was added and incubated for 30 min at 37 ° C. The plate was washed three times with distilled water and allowed to dry. The samples were observed under a fluorescence microscope and the TCID 50 / ml number was determined.

Animals:

Thirty-one pigs of both sexes and six weeks of age were obtained from dr. Kenta Schwartze. Pigs were randomly assigned to four pens by weight on day 0.

-14EN 295933 B6

Location of animals

Four pens in a small infirmary, each separated by at least 90 cm, were used to house the animals. The pens had a wire floor and were separated by a fixed partition. The space was heated with additional heating with heating lamps. The temperature was maintained between 26 and 29 ° C throughout the duration of the study.

Feed and water:

Animals were fed ad libitum with 19% protein, consisting of crushed maize and antibiotic-free soy, in stainless steel troughs. Water was also administered ad libitum.

Swine infection:

On day 0, pigs were weighed and blood samples collected by retro-orbital sinus capillary. Serum was collected and stored frozen at -20 ° C. Feces swabs were also collected for PCR. The pigs received 10 ml of inoculum administered by gavage.

Treatment Number of pigs

Control - uninfected cells5

Control - no treatment2

High doses8

Medium dose8

Low dose8

The pigs were weighed on days 0, 10, 17-24 and blood samples were taken.

Polymerase chain reaction:

Swine infection was monitored by PCR using primers and cycling parameters as described in Jones (1993). Fecal samples collected on days 0, 7, 14, 21 and 24 as well as intestinal mucosa were tested by PCR.

Histopathological examination:

Sections of the ileum, jejunum and column were fixed with formalin, processed conventionally, stained with ilematoxylin and eosin, impregnated with silver and evaluated. Sections were also stained using a monoclonal antibody specific to Z. intracellularis.

Results:

Clinical symptoms:

Clinical signs of loose stools were first observed after three days at high doses. Symptoms peaked at fourteen days and then began to differentiate.

Weight gain:

Mean daily weight gains were calculated to show that the high and medium dose group had reduced weight gains compared to the control group. When comparing the groups in terms of weight gain, the effect of dose titration was observed.

PCR:

No stool loss was observed within 14 days. After 21 days, 37.5% of high dose pigs were positive in faeces by PCR. After necropsy, intestinal mucosa was tested by PCR with 100% positive at high dose, 75% at medium dose, 50% at low dose and 0% at control.

-15 GB 295933 B6

Macroscopic damage:

Macroscopic lesions were found in two high-dose pigs (# 50 and # 202). The pigs had approximately 90 cm thickening of the intestines, in the case of # 202 with necrosis.

Histopathological examination:

FA:

Staining of FA sections of ileum, jejunum and column revealed the presence of L. intracellularis at 87.5% at the high dose, 62.5% at both the medium and small dose, and 0% at the controls.

Microscopic damage:

Damage was observed in 100% of the high dose, 75% in the middle dose, 87.5% in the low dose, and 14% in the controls. These lesions were determined by observing an increased amount of mononuclear cells in the lamina propria and the mucosal layer, often associated with Peyer's Patchers hyperplasia. Crypt hyperplasia was also observed.

Silver coloring:

Sections were stained for the presence of intracellular curved bacteria. In 87.5% of the high dose, 62.5% of the intermediate dose, and 87.5% of the low dose, the presence of bacteria was observed, whereas it was not observed in controls.

Discussion:

The pigs were successfully infected with pure L. intracellularis cultures. At doses of 10 ⁷ bacteria, 100% of the pigs showed infection by PCR and microscopic lesions. The severity of the damage and the amount of bacteria in the tissue sections was relatively low. This study is a satisfactory model of test administration of L. intracellularis due to the presence of L. intracellularis and microscopic lesions in the pig. Damage may be increased by the second dose, seven days after the first dose.

Example 5

Testing vaccine efficacy in hamsters

Target:

The aim of the experiment was to find a laboratory animal model for the determination of the safety and efficacy of avirulent live L. intracellularis vaccine in hamsters.

Summary:

A study on 40 hamsters was performed by vaccinating three-week hamsters with pure cultures of a highly passaged L. intracellularis strain and given by challenge 22 days after vaccination with pure cultures of low passage virulent material. Hamsters were divided into three groups. Group A was vaccinated with a single dose of L. intracellularis strain N72994 on day 0. Group B was designated as a control group and was not vaccinated. Both groups received two test doses of a pure culture of L. intracellularis strain N343 on days 22 and 25 after vaccination. Group C received a dose of test strain NI01494 to compare relative virulence to strain N343. Group A and B each contained 15 hamsters and group C contained 10 hamsters. Dose determination results infect 50% of tissue cultures (TCID ₅₀ ) showed that hamsters were vaccinated with 10 ⁵ TCID ₅₀ / dose. The test dose of N343 contained 10 ^{5 -5} TCID ⁵⁰ / dose. The test dose for group C was 10 ^{2 75} TCID ₅ o / dose. Faecal swabs were collected on days 0, 7, 14, 21, 29, 36 and 43 for polymerase chain reaction (PCR) testing. On day 21, 5 animals were sacrificed each of groups A and B for testing the mucous membranes by FA, hematoxaline and eosin staining and silver staining of intestinal sections to determine the persistence of bacterial settlement

-16GB 295933 B6 in vaccinated hamsters. The remaining animals were sacrificed 21 days after challenge and examined similarly.

PCR results showed the presence of intracellularis in the intestinal mucosa of 100% group A hamsters 21 days after vaccination. Group B was negative in all cases 21 days after vaccination. 21 days after challenge, 50% of hamsters were PCR positive in Group A and 100% positive in Group B. Histopathological examination of sections showed mild to severe damage in 50% of the animals in Group A and mild injury in 50% of the cases in Group B, 21 days after administration. No animals were injured 21 days after vaccination. Group C had no damage 21 days after challenge. FA and silver staining was unable to detect the presence of Z. intracellularis in any section.

It can be concluded that hamsters vaccinated with a highly passaged L. intracellularis strain showed a 50% reduction in infection when tested by PCR. The intestines were populated with a low number of intracellular organisms as shown by the absence of observed organisms in sections stained with FA and silver staining. Hamsters in Group C were unable to achieve an outbreak of infection during the study most likely due to a low dose of bacteria.

Materials and methods:

Description of hamsters:

Harlan Sprague Dawley 40 week old female hamsters were used.

Inoculum growth:

Vaccine culture:

A continuous culture of intracellularis growing on HEp-2 cells for 29 weeks was used. The culture was grown in a similar manner to that described in the test culture section except that the new HEp-2 cells were transferred to the culture every two to three weeks.

Test cultures:

One normal 75 cm ² tissue culture flask was seeded with 3.75 χ 10 ⁵ McCoys cells in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and allowed to grow for 18-24 hours at 37 ° C in an atmosphere with 5% CO ₂ . The medium was removed from the cells and 1 vial of N343 MSC X, diluted with 14 ml DMEM / 2% FBS, was added to the flask. The culture was placed in a gas incubator, evacuated and the gas replaced with a mixture of hydrogen and carbon dioxide to give a mixture of 8.0% O ₂ , 8.8% CO ₂ and 83.2% H ₂ . The culture was grown for 6 days, then the cells were scraped into a 100 ml spinner bottle with 90 ml DMEM / 2% FBS and 0.01 g Cultisphere-G beads. The culture was grown at the gas concentration indicated above. The volume of the bottle was increased by doubling the volume of the medium at weekly intervals. The culture was grown for 25 days in a rotary flask to a final volume of 250 ml.

Strain NI 01494 was grown in the same manner as strain N343.

Isolation of cultures:

Vaccine culture:

The culture was isolated by centrifugation for 20 min at 3000 x g. The biomass was resuspended in sucrose-phosphate-glutamate (SPG) solution with 10% FBS and passed through a 25 gauge needle four times. The inoculum was diluted to a final volume (15 ml) SPG / 10% FBS. .

Test cultures:

The culture was isolated by centrifugation for 20 min at 3000 x g. The biomass was resuspended in sucrose-phosphate-glutamate (SPG) solution with 10% FBS and passed four times through a 25 gauge needle. Inoku-17EN 295933 B6 lum was diluted to final volume (20 ml for strain N343 and 10 ml for strain N101494) SPG / 10% FBS.

Dosage:

Vaccine:

On day 0, all hamsters in Group A were orally vaccinated with 1 ml of the prepared vaccine.

Test administration:

10 days after vaccination, 10 group A hamsters and 10 group B hamsters received an oral dose of 0.5 ml N344 strain culture. Group C received 0.5 ml test culture of strain N101494.

Determination of the number of Intracelullaris IS:

The determination of the number of viable IS intracellularis was performed by determining the dose infecting 50% of the tissue culture (TCID ₅₀ ). This was done by withdrawing 2 ml culture for testing and lysing the cells four times by passing a 22 gauge needle. The sample was serially diluted 1:10 in DMEM / 5% FBS containing vancomycin (100 pg / ml) and amphotericin B (2.0 pg / ml). ml). Dilutions were dispensed at 0.1 ml / well into a 96 well microtiter plate, which was inoculated with 1250 cell McCoys cells per well and incubated for 18-24 hours at 37 ° C and 5% CO ₂ prior to infection. 12 wells per dilution were used. Plates were incubated for 6 days at a gas concentration of 8.0% O ₂ , 8.8% CO ₂ and 83.2% N ₂ . On day 6, cells were fixed with cold 50% acetone and 50% methanol for 2 min. 0.03 ml / well of anti-IS intracelullaris monoclonal antibody diluted 1: 2000 with PBS was added to the wells. The plate was incubated at 37 ° C for 30 min and then washed three times with PBS. Anti-mouse FITC diluted 1: 30 at 0.03 ml / well was added and incubated for 30 min at 37 ° C. The plate was washed three times with distilled water and allowed to dry. The samples were observed with a fluorescence microscope and a TCID of ₅₀ / ml was determined.

Hamster infection monitoring:

Hamster infection was monitored by PCR using primers and cycle parameters as described by Gaia Jones. Faecal samples were taken on days 0, 7, 14, 21, 29, 36 and 43 after vaccination. After the hamsters were killed, the intestinal mucosa was also tested by PCR.

Histopathological examination:

The sections of the ileum and the column were fixed with formalin, processed conventionally, stained with hematoxylin and eosin, impregnated with silver and evaluated. Sections were also stained with a monoclonal antibody specific for L. intracellularis.

Average daily weight gains:

Hamster weights were determined 21, 28, 35 and 42 days after vaccination to determine the average daily weight gain.

Results:

The results are shown in the following table.

TQD ₅₀ :

TCID50 results indicated that vaccinated group (Group A) received 10 ⁴ 'TQD50 ⁸⁶ / hamster. Hamsters in groups A and B were given test doses of strain N343 at 10 ^{5 -5} TQD50. Group C received a test compound in an amount strain N101494 10 ^{2 '75} TCBD ₅₀ / hamster.

-18EN 295933 B6

PCR:

PCR testing showed the presence of intracellularis in 100% of vaccinated hamsters who were sacrificed 21 days after vaccination. Testing 43 days after vaccination showed that 100% of the control hamsters and 50% of the vaccinated hamsters were infected with L. intracellularis. No 5 of the hamsters receiving the test dose of NI 01494 were positive. No faecal excretion of bacteria was detected during the study.

Histopathological examination:

Staining Η & E showed no histological lesions in all sections of hamsters killed at 21 days post-vaccination. In sections taken 43 days after vaccination, 50% of the vaccinated group had mild to severe lymphocytic enteritis and 50% of the control group had mild lymphocytic enteritis. No injury was observed in the N101494 test dose group. FAs did not detect L. intracellularis in any of the hamsters 43 days after vaccination.

Discussion:

Hamsters vaccinated with a highly passaged L. intracellularis strain were observed to have a 50% reduction in infection when tested by PCR.

-19EN 295933 B6

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-20EN 295933 B6

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-21 GB 295933 B6

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-22EN 295933 B6

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-23EN 295933 B6

Example 6

Investigation of vaccine efficacy in pigs

Target:

The aim of this study was to evaluate the safety, persistent bacterial population, and efficacy of avirulent live isolate and killed L. intracellularis isolate in pigs aged two to three weeks. A host animal study was conducted in which pigs were vaccinated at three weeks of age and then exposed to a virulent test dose of L. intracellularis strain N343 to compare the differences in protection between vaccines.

Methods:

On December 11, 1995, a total of 45 pigs at the age of three weeks were purchased from tří & K Farms. The pigs were transported to Veterinary Resources, Inc., located near Cambridge, Iowa, where they were identified to identify each individual. The pigs were in this research station two days before the experiments began to allow acclimatization at the station and were fed an antibiotic-free diet during the study.

On December 13, all pigs were weighed, sampled for serum, clinically examined, and rectal swabs collected. The pigs were then randomly divided into groups of five and housed in pens. Twenty pigs were placed in a separate room and designated the control group and the strict control group. Fifteen pigs were placed in the second room for ISi-1 vaccination. In the third room there were ten pigs for ISi-2 vaccination.

A live vaccine was prepared by NOBL Laboratories Research and Development and identified as the ISi-1 experimental series. ISi-1 (strain N343) was isolated from pig and allowed to grow continuously in pure culture for 29 weeks. The vaccine was grown on McCoys cells in rotary flasks at reduced oxygen until approximately 100% infection was detected. The high passaged N343 sample used for ISi-1 was grown for an additional 11 weeks ("N343NP40wk") and deposited in accordance with the Budapest Agreement 22.5. 1996 at ATCC, 12301 Parklawn Drive, Rockville, Maryland, USA 20852 and designated deposit number 55783. Cultures were isolated by centrifugation at 3000 x g for 20 min. The biomass was resuspended in a sucrose-phosphate-glutamate (SPG) solution with 10% FBS and passed through a 25 gauge needle four times. The lysates were centrifuged at 500 x g for 5 min to remove cell debris and microcarrier beads. The supernatant was stored and stored at -70 ° C until approximately one hour prior to vaccination when stored on ice until administration.

The killed vaccine (ISi-2) was grown, passaged for 12.5 weeks, and isolated in a similar manner as above and purified by a percent gradient. Purified bacteria were stored at -70 ° C until approximately 1 week prior to vaccination, when stored at 4 ° C and normal atmospheric oxygen concentration, which is toxic to L. intracellularis. A1OH was added to the bacteria until the final composition was 10% AlOH. Protein concentration was determined by the biuret method.

Determination of the amount of living IS intracellularis:

Determination of viable L. intracellularis bacteria was performed by determining the dose for infection of 50% tissue culture (TCID ₅₀ ). 96-well microtiter plates were seeded with 1250 cell / well McCoys cells and allowed to grow 18-24 hours prior to infection. Samples were serially diluted 1:10 with DMEM / 5% FBS containing vancomycin (100 µg / ml) and amphotericin B (2.0 µg / ml). Diluted samples were added to 96 well microtiter plates at 0.1 ml / well. 12 wells per dilution were used. The plate was incubated for 6 days at 37 ° C and a gas concentration of 8.0% O ₂ , 8.8% CO ₂ and 83.2% N ₂ . Cells were fixed with cold 50% acetone and 50% methanol for two minutes. 0.03 ml / well of anti-L. intracellularis monoclonal antibody (developed by Dr. Steven) was added to the wells

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McOrist), diluted 1: 2000 in PBS. The plate was incubated for 30 minutes at 37 ° C and then washed three times with PBS. Anti-mouse immunoglobulin G-fluorochrome (FITC) conjugate diluted 1: 30 at 0.03 ml / well was added and the plates incubated for 30 min at 37 ° C. The plate was washed three times with distilled water and allowed to dry. The samples were observed under a fluorescent microscope and TCID ₅₀ / ml values were determined using the Reed-Meunsch calculation method.

TCID 50 values showed that ISi-1 had 1.8 x 10 ⁵ bacteria / ml. The fourth inoculum was a placebo and was derived from tissue culture cells treated in the same way as the vaccines.

The killed vaccine was tested by the biuret method for a total protein content of 0.311 mg / ml.

The pigs were vaccinated on December 13, 1995. A live vaccine was administered to all at a dose of 2 ml intranasally of 1 ml in each nostril. ISi-2 (killed) was administered IM at a dose of 1.5 ml per pig and again 14 days later. All control animals received uninfected cells in the same way as live vaccines.

Observations and samples:

Faecal swabs and serum samples were collected at seven-day intervals throughout the study. Faecal swabs were processed for PCR testing using the primer set 5-TATGGCTGTCAAACACTCCG-3 'and 5-TGAAGGTATTGGTATTCTCC-3' for DNA amplification. The cycle parameters were 93 ° C for 5 minutes, 55 ° C for 45 seconds and 72 ° C for 45 seconds for the first cycle. 33 cycles were performed at the above temperatures with 45 seconds for each temperature. The last cycle was 93 ° C for 45 seconds, 55 ° C for 45 seconds, and 72 ° C for 2 minutes, with primers defined by Jones et al.

Administration of test microorganism:

All animals, except for strict controls, received a test dose of culture 26 and 27 days after vaccination, consisting of low-passaged cultures of L. intracellularis strains N343 and N72994, which were grown continuously between 8 and 12 weeks. Cultures were isolated by centrifugation at 3000 x g for 20 min. The biomass was resuspended in sucrose-phosphate-glutamate (SPG) solution with 10% fetal bovine serum and passed four times through a 25 gauge needle. Some isolated cultures were stored at -70 ° C until administration, while others were grown until the day of administration and then isolated. Test inoculum was pooled and TCID 50 cultures were determined. The samples were stored until administration on ice.

The test culture was administered on January 8, 1996 and contained 4 x 10 ⁴ bacteria / ml and the test culture administered on January 9, 1996 contained 3 x 10 ⁴ bacteria / ml. The pigs were gavaged on a 15 ml test dose on both days. Thus, the animals received 6 x 10 ⁵ bacteria / pig and 4.7 x 10 ⁵ bacteria per pig

8 January 1996 or 9 January 1996.

Results:

Safety test:

Fecal PCR results:

Detection of L. intracellularis by PCR showed that no pigs were secreted by the bacteria at the start of the study. Seven days after vaccination, all animals were negative. Fourteen days after vaccination, three pigs in the ISi-1 group were positive. In the ISi-1 group, two animals were positive 21 days after vaccination and all other pigs were negative. On the 26 th day after vaccination, no animals were secreted by PCR detection. 26 days after vaccination, five ISi-1 and control pigs and four ISi-2 pigs were sacrificed. Samples of ileum, colon, mesenteric lymph nodes and tonsil were collected as well as lung samples in pigs suspected of having pneumonia.

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PCR was performed on individual ileum and lung samples. Tonsils, columns and lymph nodes were pooled for each group and PCR was performed. PCR test results are shown below.

Group PCR ilea 26 days after vaccination Sfiojený columns United tonsily United mezenter. Ivmf.uzlinv Connected lungs ISi-1 1 out of 5 + * 1 of 1 ISi-2 0 out of 4 - - - 0 out of 1 Checks Oz 5 - netest Strict controls netest netest. netest netest. netest.

Histological sections of the ileum were stained using a monoclonal antibody specific to L. intracellularis as the primary antibody and an anti-mouse immunoglobulin G-fluorochrome conjugate as the secondary antibody. L. intracellularis was observed in three out of five ISi-1 pigs. Fluorescent antibody staining was negative for all other animals.

The remaining animals were sacrificed 21 days after administration of the test dose and samples were taken for evaluation. The PCR results are shown below.

Group Ileea PCR 21 days after vaccination Connected columns United tonsiiv United mezenter. lymph node Connected lungs ISi-1 0 out of 10 + - - - ISi-2 0z6 - - - - Checks 4of 10 - - - Strict controls 0z5 - * -

FA staining of the ileum was performed as above, with seven out of ten animals positive in the control group. All other animals were negative for the presence of L. intracellularis.

The serum was tested for IgG antibody production in pigs after exposure to L. intracellularis. The assay was performed by inoculating tissue culture-adapted Terasaki plates with McCoys 20 cells at 125 cells / well, which were allowed to grow for 18-24 hours prior to infection. Then she was into

A pure L. intracellularis culture, diluted to 1000-3000 bacteria / ml DMEM with 5% fetal bovine serum at 0.01 ml / well was added to the wells. The plate was incubated for 6 days at 8.0% O ₂ , 8.8% CO _2, and 83.2% N ₂ gas concentrations. Cells were fixed with cold 50% acetone and 50% methanol for two minutes. Sera for pigs were diluted 1: 75 with sterile PBS. Dilute serum was added to the wells at 0.01 ml / well. Plates were then incubated for 30 to 60 min at 37 ° C. Plates were washed five times with sterile PBS. 0.01 ml / well of immunoglobulin G anti-pig IgG conjugate - fluorochrome was added to the wells. The plates were incubated for 30 min at 37 ° C. They were then washed five times with distilled water and allowed to dry. The samples were washed five times with distilled water and allowed to dry. Samples were observed with a fluorescence microscope and wells in which bacteria were observed were marked as positive and wells in which no bacteria were observed were marked as negative.

Results:

Group Day 0 26 days after vaccination 47 days after vaccination, 21 days after challenge ISi-1 0 out of 15 6of 15 8z 10 ISi-2 0 out of 10 3 out of 10 5 z6 Checks 1 of 15 0 out of 15 9 out of 10 Strict controls 0z5 0 out of 5 Oz 5

Animals that were positive on day 0 were retested at weekly intervals. The results showed that all became serologically negative within 14 days after vaccination. This is not unexpected because the age of pigs on day 0 was three weeks and positive results at that age could be due to maternal antibodies.

Sera were tested together with positive control sera obtained by hyperimmunization of porcine L. intracellularis grown in pure culture. The negative control serum used was collected from a gnotobiotic pig at South Dakota State University.

The results above and examples are illustrative only for preferred embodiments that achieve the objects, features and advantages of the present invention and are not intended to limit the present invention. Any modifications of the invention that fall within the scope of the following claims are considered to be part of the present invention.

Claims (21)

PATENT CLAIMS A method for culturing L. intracellularis in cell culture cells in a medium, comprising the steps of incubating L intracellularis infected cell culture cells at an oxygen concentration of less than 18% of the maximum saturated oxygen concentration in the medium, wherein the cell cells cultures are kept in suspension. -27EN 295933 B6 Method according to claim 1, characterized in that the cell culture cells are maintained in suspension for 6 to 8 months. The method according to claim 1 or 2, characterized in that, to increase the production of L. intracellularis, it comprises an additional step wherein part of the cell culture cells infected with L. intracellularis is passaged into fresh uninfected cell culture cells. 4. The method of claim 3, further comprising the step of isolating at least a portion of the cell culture cells. The method of claim 1, wherein the L. intracellularis bacterium is obtained from an animal infected with Z. intracellularis. 6. The method of claim 5 wherein the animal is a pig. The method of claim 1, wherein the incubation is performed at an oxygen concentration ranging from 0% to 8% of the maximum saturated oxygen concentration in the medium. The method of claim 7, wherein the incubation is performed at an oxygen concentration of from 0% to 3% of the maximum saturated oxygen concentration in the medium. The method of any one of claims 1-8, wherein the cell culture cells are cells selected from the group of Hep-2 and IEC-18. Method according to any one of claims 1 to 8, characterized in that the incubation is carried out on McCoys and IEC-18 cells on microcarriers. The L. intracellularis bacterium deposited under deposit number ATCC 55672, produced by the method of claim 1. 12. A method for culturing L. intracellularis comprising the steps of: (1) inoculate cell culture cells with an inoculum containing L. intracellularis to infect the cells of the bacteria; and (2) the infected cells are incubated at a temperature of 36 ° C to 38 ° C at an oxygen concentration of 0% to 8% of the maximum saturated oxygen concentration in the medium while stirring the infected cell culture cells such that cultivation of L. intracellularis bacteria is maintained infected cell culture cells in suspension. 13. A method for producing an attenuated strain of L. intracellularis comprising recovering cell culture cells infected with L. intracellularis, incubating said infected cell culture cells at an oxygen concentration of from 0% to 18% of the maximum saturated oxygen concentration in the medium, mixing said infected cell culture cells such that the culturing of the bacteria proceeds while keeping the infected cell culture cells in suspension, passaging at least a portion of the cultured bacterium, isolating at least a portion of the cultured bacterium, and selecting an attenuated strain to recover the attenuated L. intracellularis. Use of an L. intracellularis bacterium cultured by the method of any one of claims 1 to 10, or an antigenic component thereof, as an antigen in a diagnostic assay for detecting the presence of antibodies that specifically react with L. intracellularis in a biological sample. Use according to claim 14, wherein the assay is an ELISA or Western blot or immunoassay, preferably an immunofluorescence assay. -28EN 295933 B6 16. A method for determining the presence of antibodies specifically reacting with L. intracellularis in a biological sample, comprising the steps of obtaining cell culture cells infected with L. intracellularis, incubating the infected cell culture cells at an oxygen concentration of from 0% to 18% of maximum saturated concentration of oxygen in the medium, mixing said infected cell culture cells to cultivate L. intracellularis while keeping the infected cell culture cells in suspension, isolating at least a portion of the cultured L. intracellularis bacterium, recovering the biological sample from the animal, contacting the recovered sample with the isolated L. intracellularis or a component thereof derived from it under conditions in which the antibody present in the biological sample reacts with L. intracellularis or a component thereof and determining whether an antibody-antigen reaction has occurred and thereby determining presence of an antibody against L. intracellularis in the sample. 17. A vaccine for eliciting an immunological response against L. intracellularis in an animal comprising an attenuated L. intracellularis strain produced by the method of claim 13, in a pharmaceutically acceptable carrier, wherein the attenuated strain is in an effective amount to elicit an immunological response. A vaccine for eliciting an immunological response against L. intracellularis in an animal comprising killed L. intracellularis in a pharmaceutically acceptable carrier, wherein the bacterium is produced by the method of any one of claims 1 to 10 and is in an amount effective for inducing an immunological response. 19. The vaccine of claim 17 or 18, wherein the L. intracellularis infecting cell culture is prepared from the intestinal homogenate of an animal infected with L. intracellularis. 20. The vaccine of claim 17, wherein the attenuated strain is N343NP40wk. The attenuated strain deposited under Deposit Number ATCC 55783, produced by the method of claim 13.